JP3160288B2 - Purine nucleosides - Google Patents

Abstract

There are disclosed nucleosides having Formula I: <IMAGE> I wherein R is H or acyl. These compounds have anticancer activity.

Description

Description: BACKGROUND OF THE INVENTION This invention relates to certain 2'-F, 2-halo substituted purine nucleosides.

Certain arabinoflabinosyl nucleosides have known antiviral (araA) and anticancer (araC) activity. Arabinofuranosyl nucleosides having a 2'-substituent other than a hydroxyl group also show useful biological effects. All of these nucleosides require activation (phosphorylation) to be effective, and this is generally done by enzymes different from the corresponding ribofuranosyl nucleosides.

U.S. Patent Application No. 4,751,221, such as Watanabe, has the following formula: Wherein X and Y are the same or different;
Hydrogen, OR ³ (keto or enol), SR ³ , NR ³ R ⁴ , NH acyl or halogen such as chlorine or bromine.

R ³ and R ⁴ are the same or different and include lower alkyl having 1 to 7 carbon atoms such as hydrogen, methyl, ethyl, propyl and the like, benzyl, benzhydryl, p-
Aralkyl such as methoxybenzyl, or phenyl,
Aryl such as p-chlorophenyl, toluyl, p-methoxyphenyl, naphthyl and the like.

NH acyl can be alkanoyl or aroylamide, wherein R ¹ and R ² are the same or different hydrogen or formyl,
Acetyl, propionyl, isopropionyl, butyryl, isobutyryl, tertiary butyryl, valeryl, pivaloyl, caproyl, capryl, lauryl, myristyl,
An acyl group which can be an alkanoyl group having 1 to 20 carbon atoms, such as palmityl, stearyl, arachidyl, styridyl, palmitoleyl, oleyl, linolenyl, arachidonyl and the like. ] The nucleoside shown by these is disclosed.

Montgomery et al., “Journal of Medicinal
Chemistry, 1986, Vol. 29, No. 11, pp. 2389-2392 has the following formula: Wherein R ¹ and R ² are the same and can be Cl or NH ₂ , R ³ can be benzoyl or hydrogen, and R ⁴ can be acetyl or hydrogen. ] The nucleoside shown by these is disclosed.

(Summary of the Invention)

Incorporating a 2-halo substituent into the purine ring significantly alters the metabolism of adenine nucleosides, specifically by reducing the ability of the compound to be a substrate for adenosine deaminase, in the arabino configuration. It has been reported that substituting fluorine for C-2 'renders these derivatives highly resistant to phosphorolytic cleavage, and that the combination of these two moieties in the same molecule confers anticancer activity. Has been found in.

Thus, in the present invention, Formula I, Wherein Z is F, Cl, or Br, and R is hydrogen or acyl. ] The nucleoside shown by this is provided. When R is acyl, this derivative acts as a prodrug to prolong the body life of the parent drug.

The compounds of this invention are useful for treating cancer, for example, chronic lymphocytic leukemia.

[Detailed Description of the Invention]

In the discussion below, reference is made to Equation 1 below.
The examples also include descriptions of compounds in which the various substituents in these compounds are represented by 1a-1g as defined in Formula 1 below.

In the definition of these substituents, "Bz" means benzoyl and "Ac" means acetyl.

 The following example illustrates the preparation of a compound of Formula 1.

Example 1 2,6-dibromo-9- (3-O-acetyl-5-O-
Benzyl-2-deoxy-2-fluoro-β-D-arabinofuranosyl) -9H-purine (1a) 3-acetyl-5 in 400 mL of dry dichloroethane
The benzoyl-2-deoxy-2-fluoro-arabinofuranosyl bromide (33.2 mmol) solution was stirred with 4A molecular sieve for 10 minutes before 2,6-dibromopurine (9.3 g, 33.5 mmol) was added. The mixture was vigorously stirred with an elevated stirrer and placed in a preheated 100 ° C. oil liquid bath. Heating was continued for 32 hours until all the bromo sugar was consumed.
(4- (4-nitrobenzyl) pyridine for detection
The mixture was cooled to room temperature using a spray, TLC 2: 1 cyclohexane-ethyl acetate) and filtered through celite. The solid was washed with dichloroethane and the combined filtrate was evaporated in vacuo to dryness. The residue (16.5 g) was separated by flash chromatography on 150 g silica gel (230-400 mesh) using 2: 1 cyclohexane-ethyl acetate as eluent3.
A mixture of different nucleosides. By combining the pure fractions, 3.64 g of a chromatographically homogeneous but non-crystallizing glass (19.
7%). A second column made on the impure fraction gave 2.21 g (11.9%) of a purer product for a total yield of 31.6%. MS 557 (M + 1) ⁺ . Elemental analysis:
Calcd for _{C 19 H 15 Br 2 FN 4} O 5 · 0.2C 6 H 12: C, 42.19;
H, 3.05; N, 9.74. Found: C, 42.11; H, 3.10; N, 9.35.

Example 2 2-bromo-9- (2-deoxy-2-fluoro-β-
D-arabinofuranosyl) -9H-purin-6-amine (1b) Stainless steel lined with 400 mL of a solution of 1a (5.84 g, 10.5 mmol) in ethanolic ammonia (saturated at 0 ° C.) And placed at room temperature for 3 days. The solution was evaporated to dryness and evaporated with ethanol to remove ammonia. The residue containing the desired material and the 5'-benzoyl compound was dissolved in 440 mL of acetonitrile and 120 mL of water. Lithium hydroxide monohydrate (881 mg, 21 mmol) was added and the solution was stirred at room temperature for 16 hours. Thin layer chromatography (5: 1C
HCl ₃ -MeOH) indicated the completion of the reaction. The cooled solution was carefully neutralized with glacial acetic acid and evaporated to dryness. The white solid residue was recrystallized from water. The product was dried in vacuo and at room temperature at 100 ° C. for 2 hours. 2.15 g (59.2%). Mp209-2
10 ° C., TLC 5: 1 CHCl ₃ -MeOH R _f 0.47; HPLC 99.8%
9: 1; H ₂ O-MeCN; MS, 348 (M + 1) ⁺ ; UV λmax pH
1 264 (14.3), pH 7 264 (14.9), pH 13 264
(15.2). Elemental analysis: Calculated for C ₁₀ H ₁₁ BrFN ₅ O ₃ :
C, 34.50; H, 3.18; N, 20.12; experimental: C, 34.36; H, 3.28; N, 19.
93. Example 3 2-chloro-9- (2-deoxy-2-fluoro-β-
D-arabinofuranosyl) -9H-purin-6-amine (1d). Montgomery et al., "Journal of Medicinal Chemistry", 1986, Vol. 29, No. 11, 2389-2392.
A solution of 1c (5.1 g, 10.9 mmol) in ethanol (100 ° C.) saturated with anhydrous ammonia (100 mL), prepared according to the method described on page 5, in a glass-walled stainless steel bomb, Placed at room temperature for 3 days. TLC (2: 1 cyclohexane-ethyl acetate and 5: 1
CHCl ₃ -MeOH) showed that no starting material. However, there were two main products. That is, the target compound and its 5'-benzoyl analog. The solution was evaporated to dryness and co-evaporated with acetonitrile.
The residue was dissolved in acetonitrile (100 mL) and water (60 m
L) and diluted with lithium hydroxide monohydrate (915mg, 21.8mmo
l) was added. The solution was stirred for 3 hours at room temperature, but when _{TLC (5: 1 CHCl 3 -MeOH} ) showed complete reaction. The solution was cooled, neutralized with acetic acid and evaporated to dryness. Pure compound was obtained after three recrystallizations from water. 14.g (42.3
%). Mp 225-226 ° C; TLC 5: 1 CHCl ₃ -MeOH, R _f 0.
40; HPLC 99% 4: 1 H 2 O-MeCN; Ms, z / e 304 (M +
1) ⁺ ; UV λmax 263 (15.5) at pH 1; 263 (16.
2); 263 at pH 13 (16.4). Elemental _{analysis: C 10 H 11 ClFN 5 O} 3 · H 2 O
Calculated for: C, 37.34; H, 4.07; N, 21.77. Experimental value: C, 3
7.62; H, 3.98; N, 21.88.

Example 4 2-Fluoro-9- (3-O-acetyl-5-O-benzoyl-2-fluoro-β-D-arabinofuranosyl)
-9H-purin-6-amine (1f). Diamino compound 1e, (700 mg, 1.63 mmol) produced according to the method described in Montgomery et al., "Journal of Medicinal Chemistry", 1986, vol. 29, no. Dissolved in 3: 2 HF-pyridine (15 mL) and tertiary butyl nitrile (271 μl,
2.28 mmol). After 1 hour at -20 ° C, TLC showed the reaction was incomplete. Tertiary butyl nitrile (70 μl, 0.59 mmol) was further added, and the reaction was quenched.
Continued at 20 ° C. for another 2 hours. Saturated cold reaction solution containing ice
NaHCO ₃ (1 mL) was added dropwise. The foaming mixture was stirred vigorously for 20 minutes and then diluted with CHCl ₃ (300 mL). The layers were separated and the aqueous layer was extracted with more CHCl ₂ (2 × 175 mL). The combined organic extracts were washed with water (3 × 175 mL), dried (MgSO ₄ ) and evaporated to dryness. Using silica gel in CHCl ₃ to CHCl ₃ as the eluent (230-400
Mesh). Fractions were combined to give essentially pure If, 500 mg (70%). EtOH
Pure (1f) was obtained by crystallization of a small sample from. Mp 208-209 ° C; TLC 95: 5 CHCl ₃ -MeOH, R _f 0.4
5; HPLC 99%, 1: 1H 2 O-MeCH; MS, z / e 434 (M +
1) ⁺ . Elemental _{analysis: C 19 H 17 F 2 N} 5 O 5 Calcd for: C, 52.6
6; H, 3.95; N, 16.16. Found: C, 52.48; H, 3.92; N, 15.98.

Example 5 2-fluoro-9- (2-deoxy-2-fluoro-β
-D- Arabinofurashiru) -9-H- purin-6-amino (1 g) 1: suspension of 1MeCN-H ₂ O (40mL) solution of 1f (430 mg, 0.99 mm
ol) with solid lithium hydroxide monohydrate (125mg, 2.97mmol)
Was treated at once. After stirring at room temperature for 20 minutes, the reaction became a clear solution. The 3-h TLC moiety indicated that deprotection was complete. Glacial acetic acid (57 μl) was added and the solution was evaporated until a white solid precipitated. After cooling, the solid was collected, washed with cold water, dried in vacuo at room temperature, crude 1 g, 252
mg was obtained. This solid was dissolved in 40 mL of water, and the water balance SM-4 was added.
Bio-bead columns (1.5 x 32 cm) were applied. After the first elution with water, the product is subjected to a step gradient: 5% in water-
--Eluted with 20% EtOH. The combined and evaporated residue from the column fraction is crystallized after charring from 25 mL of boiling water,
Dried in vacuum at 56 ° C. for 16 hours, pure 1 g, 178 mg (59 mg
%). Mp 207-209 ° C; TLC 5: 1 CHCl ₃ -MeOH,
_{R f 0.50; HPLC 99%,} 9: 1 H 2 O-MeCN; MS, z / e 288
(M + 1) ⁺ ; UV λ max pH 1 261 (14.0), 268
(Sh), pH7 260 (15.1), 268 (sh), pH13 261 (1
4.9), 268 (sh). Elemental _{analysis: C 10 H 11 F 2 N} 5 O 3 · H 2 O Calculated for: C, 39.25; H, 4.29 ; N, 22.94. Experimental values: C, 39.51; H,
4.21; N, 22.94.

If rapid clearance is an issue, it may be desirable to prepare 5'-O-acyl derivatives of the compounds of Examples 2, 3 and 5. Such a 5'-O-acyl derivative is a prodrug having an increased activity based on an increase in half-life in living tissue, and can carry a more active drug to a target site. . The general experimental procedure for 5-acylation is as follows.

Example 6 Dry 2-halo-9- (2-deoxy-2-fluoro-β in 10 mL of 1: 1 pyridine-N, N-dimethylformamide
A solution of -D-arabinofuranosyl) adenine (1 mmol) was cooled to 0 ° C and treated dropwise with the appropriate acyl chloride (1.25 mmol). After 6 hours at 0 ° C, the solution is ice-water (100 mL)
, Stirred for 15 minutes and frozen overnight. The collected white solid precipitate was washed with cold water, dried at room temperature under vacuum,
The crude 5'-acylated nucleoside was obtained. If no precipitate formed, the reaction mixture was extracted with CHCl ₃ (4 × 25 mL). The combined extracts were dried (MgSO ₄ ) and evaporated to give the crude 5′-acylated nucleoside as a glass.
If necessary, the crude product 9 was purified by silica gel chromatography on a flash column or thin layer as eluent CHCl ₃ -MeOH 1. Pure 5'-acylated nucleosides were obtained as crystalline solids from a suitable recrystallization solvent such as acetonitrile, diethyl ether, or toluene-acetonitrile.

Example 7 Comparison of cytotoxicity Table 1 shows the results of the cytotoxicity test. IC for L1210 cells, CCRF-CME cells, and K562 cells
₅₀ is the concentration required to reduce cell growth by 50% compared to untreated controls. Cells were grown in suspension culture and the number of cells present was determined at 24, 48 and 72 hours. The values shown in Table 1 are for 48 hours for L1210 cells and 72 hours for CCRF-CEM cells and K562 cells.

The IC ₅₀ for H.Ep.-2 cells is the concentration required to reduce colony formation by 50% compared to controls. 100 cells in 10 mL of medium were placed in a prescription bottle and after 10 days of incubation, the medium was gently decanted and the colonies were stained and counted.

Example 8 Summary of Anticancer Activity CD2F1 mice were inoculated with 10 ⁶ p388 white blood cells intraperitoneally (ip). The day of tumor implantation was indicated as day 0. Compounds were administered intraperitoneally at several dose levels, from toxic to non-toxic. Control mice with tumors were not treated.
The mice were observed during their lifetime. Anticancer activity was assessed based on the percentage increase in median survival (% ILS) and effective log cell death. The calculation of the effective log cell death is calculated as
Performed from tumor doubling time determined from internal tumor titers consisting of implants from −10 dilution. Long-term survivors are ILS
And was excluded from the percentage of tumor cell death. To assess tumor cell death at the end of treatment, differences in survival time between treatment and control groups were adjusted to account for the regenerating length of the tumor cell population that could occur during each treatment. Table 2 shows the results.

Example 9 This example demonstrates the superiority of the compounds of this invention over the aforementioned anti-cancer compounds, such as 2-fluoro-9-β-D-arabinofuranosyl adenine, ie, which are not so rapidly degraded by the enzyme purine nucleoside phosphorylase. Shows sex. Purine nucleoside phosphorylase was partially purified from E. coli B (lyophilized cells) obtained from Sigma Chemical Company. The enzyme reaction mixture consisted of nucleoside substrate (0.5 mM), phosphate buffer (50 mM, pH 8.0) and enzyme in a final volume of 1.0 mL. The reaction was terminated after 30, 60, 120, 180 and 240 minutes incubation and the amount of nucleosides and bases present was determined by HPLC on a reversed phase column. The results are shown in Table 3.

────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Seek List, John A., The Third United States 35242 Alabama, Birmingham, Sally Lane No. 5564 (56) References JP-A-1-149797 (JP, A Japanese Translation of PCT Application No. 3-501258 (JP, A) The Journal of Organic Chemistry, Vol. 34, no. 9 (Septmber 1969) p. 2632-2636 (58) Fields investigated (Int. Cl. ⁷ , DB name) C07H 19/16-19/19 A61K 31/7076 CAPLUS (STN) REGISTRY (STN)

Claims (6)

(57) [Claims] (1) A compound of the following formula (I) together with a pharmaceutically acceptable carrier for administration: Wherein Z is F, Cl or Br, R ₁ is hydrogen or acetyl, and R ₂ is hydrogen or acyl. A pharmaceutical composition for anticancer treatment, comprising a compound represented by｝ as an active ingredient. 2. The method of claim 1, wherein R ₁ and R ₂ are hydrogen.
A pharmaceutical composition according to claim 1. 3. The pharmaceutical composition according to claim 1, wherein Z is Cl. 4. The pharmaceutical composition according to claim 1, wherein Z is F. 5. The pharmaceutical composition according to claim 1, wherein Z is Br. 6. A cytotoxic effect in cancer cells of a mammal, comprising an effective amount of a compound according to any one of claims 1 to 5 together with a pharmaceutically acceptable carrier for administration as an active ingredient. A pharmaceutical composition for producing.